We are developing a live-attenuated, intranasal, pediatric vaccine against human respiratory syncytial virus (RSV). Building on years of molecular and biologic studies, we use reverse genetics to produce highly defined vaccine candidates from cloned cDNAs. This provides well-defined vaccine viruses that can have improved properties. This also provides virus with a short, well-defined passage history that greatly diminishes the chance of adventitious agents. It addition, the DNA intermediate provides a stable vaccine seed as well as the means to modify the virus as necessary in response to clinical data. Five cDNA-derived viruses have been evaluated to date in clinical studies, as described in previous years. The most promising virus, called rA2cp248/404/1030/delSH, was safe and immunogenic in young infants. However, there was evidence of loss of the 248 or 1030 attenuating point mutation (Tyr-1321-Asn or Gln-831-Leu, respectively, in the L protein) in some isolates. We are continuing to work to increase the genetic stability of this virus, and expect to have an improved candidate to enter phase 1 studies in 2011 or 2012. This ongoing work will be described in next years report. We also have identified new combinations of mutations that appear to have promising properties of attenuation and stability, and which also will be reported next year. As noted in an accompanying report, another new vaccine candidate, Medi delM2-2, entered phase 1 clinical studies in August, 2011. We also have been studying RSV immunobiology. One of the signal features of RSV is that it can readily re-infect symptomatically during infancy and throughout life without the need for significant antigenic change (in contrast, for example, with influenza A virus, for which significant antigenic change is needed for re-infection). This is frequently interpreted as evidence that RSV inhibits or subverts protective immunity. This is of potential importance for vaccine development. Previously, we showed that re-infection may be aided by a viral immune evasion mechanism involving a secreted form of the attachment G protein. As another approach to this issue, we previously initiated studies of the effects of RSV and other respiratory viruses on human dendritic cells (DC) in vitro. These are potent antigen presenting cells that play a major role in initiating and modulating the adaptive immune response. We compared the effects of RSV on human monocyte-derived DC (MDDC) in a side-by-side comparison with human metapneumovirus (HMPV) and human parainfluenza virus 3 (HPIV3) using viruses that expressed green fluorescent protein (GFP) in order to monitor infection. We also included influenza A virus (Flu) in the comparison. We analyzed MDDC that were exposed to these viruses in vitro for the expression of 62 genes pertinent to maturation. One of these was CCR7, which normally is up-regulated during maturation and directs migration of antigen-bearing DC to T cell-rich zones in lymphatic tissue. We found that DCs infected with RSV or HMPV did not efficiently up-regulate CCR7, in contrast to HPIV3 and, especially, Flu. This was confirmed at the level of cell surface protein expression. In addition, HMPV and RSV did not efficiently down-regulate surface expression of CCR1, 2 and 5, which maintain DC residence in peripheral tissues and normally are down-regulated during maturation. In an in vitro migration assay, RSV- and HMPV-treated DC migrated less efficiently to the CCR7 ligand CCL19, which directs DC chemotaxis to lymphatic tissue. Secondary stimulation with lipopolysaccharide reversed this phenotype, suggesting that it is due to suboptimal stimulation rather than irreversible inhibition. This phenotype appeared to be partly due to reduced expression of pro-inflammatory cytokines by DC treated with HMPV and RSV. Inefficient migration of DC in response to RSV and HMPV infection could contribute to a dampening of the adaptive response to these viruses. We investigated whether these viruses differentially affect the activation of CD4 T cells by virus-treated DC, as has been widely hypothesized. We infected human MDDC with RSV, HMPV, HPIV3, or Flu, and compared their ability to induce proliferation of autologous CD4+ T cells in vitro. We investigated both virus-specific memory responses as well as superantigen-induced responses. In general, there was little evidence of virus-specific inhibition. There was a trend of increasing memory responses, HMPV <RSV <HPIV3 <Flu, but the differences were not significant. Overall, cytokine production by the proliferating T cells was similar among the different viruses, with no evidence of Th2 or Th17 skewing. These results provided no evidence of marked differences between the viruses in their effects on CD4 T cell activation. We recently demonstrated that the RSV NS1 protein, an antagonist of host type I interferon (IFN-I) production and signaling, also has a suppressive effect on the maturation of human dendritic cells (DC) due in part to suppression of IFN-I production. Here we investigated whether NS1 affects the ability of DC to activate CD8+ and CD4+ T cells. Human DC were infected with RSV deletion mutants lacking the NS1 and/or NS2 genes and assayed for the ability to activate autologous T cells in vitro, which were analyzed by flow cytometry. Deletion of the NS1, but not NS2, protein (i) increased the proliferation and activation of CD8+ T cells that express CD103, a tissue homing integrin that directs CD8+ T cells to the respiratory mucosa and triggers cytolytic activity (ii) increased the activation and proliferation of Th17 cells, which have recently been shown to have anti-viral effects, and (iii) skewed the Th1/Th2 balance towards Th1 by reducing the number of IL-4-producing CD4+ T cells, which are associated with enhanced RSV disease. Taken together, these data demonstrate that expression of NS1 by wild type RSV suppresses two protective cell populations (CD103+ CD8+ T cells and Th17 cells), and promotes Th2 cells that can enhance RSV disease. These studies are of particular relevance since vaccine candidates that are being developed include ones in which the NS1 or NS2 gene has been deleted. The present data suggest that a vaccine candidate lacking the NS1 gene would induce a qualitatively different immune response that might be associated with less reactogenicity and better protection.